Basolateral membrane potential and conductance in frog skin exposed to high serosal potassium

Summary In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltageV _t clamped (except for brief perturbations V _t) at zero. The skins were impaled from the outer surface with 1.5m KCl-filled microelectrodes (R _el>30 M). The transepithelial (short-circuit) currentl _i and conductanceg _t=–I _t/V _t, the outer membrane voltageV _o (apical reference) and voltage-divider ratio (F _o=V _o/V _t), and the microelectrode resistanceR _el were recorded continuously. Intermittent brief apical exposure to 20 m amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values ofg _i andF _o before and after amiloride or as the ratio of changes (–I _c/V _i) induced by amiloride. On serosal substitution of Na by K, within about 10 min,I _c declined andg _t increased markedly, mainly as a consequence of increase ing _p. The basolateral membrane voltage (V _i(=–V _o) was depolarized from 75±4 to 2±1 mV [mean±sem (n=6)], and was partially repolarized following amiloride to 5±2 mV. The basolateral conductance increased in high serosal K, as estimated by both methods. Essentially complete depolarization of the basolateral membrane and increase in its conductance in response to high [K] were obtained also when the main serosal anion was SO₄ or NO₃ instead of Cl. On clampingV _t over the range 0 to +125 mV in K₂SO₄-depolarized skins, the quasi-steady-stateV _o V _t relationship was linear, with a mean slope of 0.88±0.03. The above results demonstrate that, in a variety of conditions, exposure to high serosal K results in essentially complete depolarization of the basolateral membrane and a large increase in its conductance.     

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: Role of Ca2+-dependent K+ channels

Summary Eleven different secosteroids or steroids (10^–10 to 10^–8 m) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibianNecturus kidney while recording basolateral cell membrane potentialV _m. Seven of these molecules (1,25(OH)₂D₃, 25(OH)D₃, 24,25(OH)₂D₃, 5,6-trans-25(OH)D₃, 19-diol-cholesterol, estradiol and testosterone) resulted in changes ofV _m (V _m) occurring in a few seconds, the largest V _m being observed with 1,25(OH)₂D₃, +6.5±0.75 mV (n=19); these seven (seco)steroids but not the four inactive sterols (vitamin D₃, cholesterol, 1D₃ and aldosterone) possess a hydroxyl group on at least one carbon of the C₁₇ to C₂₅ lateral chain of the sterol ring. The V _m effect was present in Na⁺-free or Cl^–-free media, but it was abolished in HCO₃-free media. Depolarization of cell membrane potential by addition of glucose, 11mm, in luminal perfusion fluid abolished the 1,25(OH)₂D₃-evoked V _m effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K⁺ conductances, suppressed the 1,25(OH)₂D₃-evoked V _m effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca²⁺-dependent K⁺ channels also abolished the 1,25(OH)₂D₃-evoked depolarization. Such observations are consistent with the presence of Ca²⁺-dependent K⁺ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)₂D₃ and probably by other (seco)steroids.     

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Summary Cystic fibrosis (CF) is characterized by abnormal epithelial Cl^– conductance (G_Cl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected G_Cl(CF-G_Cl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though G_Cl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-G_Cl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-G_Cl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: -adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased G _tby about 2.3-fold (n = no. of ducts = 8). Removal of media Cl^–, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of G_Cl. cAMP activated both apical and basolateral G_Cl. cAMP hyperpolarized gluconate: Cl^– (lumen: bath) transepithelial bionic potentials (V _t=–20.3±5.2 mV, mean ±se, n=9) and transepithelial 3 1 luminal NaCl dilution diffusion potentials (V _t=–8.8±2.9 mV, n=5). cAMP activated basolateral G_Cl as indicated by increased bi-ionic (gluconate: Cl^–, bath: lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (V _t=–6.9±0.8 mV, n=24, nonresponsive vs. –19.4±1.8 mV, n=22, responsive ducts) but larger bi-ionic potentials (–94±6 mV, n=35, nonresponsive vs. –65±5 mV, n=17, responsive ducts) and dilution diffusion potentials (–40±5 mV, n=11, nonresponsive vs. –29±3 mV, n=7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of G_Cl in nonresponsive ducts and submaximal activation of G_Cl in responsive ducts. We conclude that cAMP activates CF-G _Cl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.Abbreviations CF cystic fibrosis - G _t transepithelial conductance - V _b electrical potential across the basolateral membrane - V _a electrical potential across the apical membrane - V _t transepithelial potential - V _b transepithelial currentinduced voltage deflections across the basolateral membrane - V _a transepithelial current-induced voltage deflections across the apical membrane - V _t transepithelial current-induced voltage deflection across the epithelium - VDR voltage divider ratio - G_Cl transepithelial Cl^– conductance - CF-G_Cl cystic fibrosis-affected Cl^– conductance - EMF electromotive force - IPR isoproterenol - IBMX 3-isobutyl-1-methylxanthine - CPT-cAMP chlorophenylthio-adenosine 3-5 cyclic monophosphate - PGE₂ prostaglandin E₂     

Intracellular sodium activity and sodium transport inNecturus gallbladder epithelium

Summary Ion-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na] _i , electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na] _i : 9.2 to 12.1mm; transepithelial potential difference, _ms : –1.5 mV (lumen negative); basolateral cell membrane potential, _es : –62 mV (cell interior negative); sodium conductance of the luminal cell membrane,g _Na: 12 mho cm^–2; active transcellular sodium flux, 88 to 101 pmol cm^–2 sec^–1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.     

Mechanisms of voltage transients during current clamp inNecturus gallbladder

Summary Microelectrode techniques were employed to study the mechanisms of the transepithelial voltage transients (V _ms ) observed during transmural current clamps in the isolatedNecturus gallbladder. The results indicate that: a) part of V _ms is due to a transepithelial resistance change (R _t ), and part to a tissue emf change. b) R _t is entirely caused by changes of the resistance of the paracellular pathway. At all current densities employed, the measured changes are probably due to changes in both fluid conductivity and width of the lateral intercellular spaces. At high currents, in addition to the effects on the lateral spaces, the resistance of other elements of the pathway (probably the limiting junction) drops, regardless of the direction of the current. c) The magnitude and polarity of the R _t -independent transepithelial and cell membrane potential transients indicate that the largest emf change takes place at the basolateral membrane (E _b ), with smaller changes at the luminal membrane (E _a ) and the paracellular (shunt) pathway (E _s ). It is shown that two-thirds of the transient are caused by E _s , and one-third by (E _b –E _a ). E _s can be explained by a diffusion potential generated by a current-dependent NaCl concentration gradient across the tissue. E _a and E _b are caused by [K] changes, mainly at the unstirred layer in contact with the basolateral membrane.     

Membrane bending energy and shape determination of phospholipid vesicles and red blood cells

A procedure is developed to calculate red blood cell and phospholipid vesicle shapes within the bilayer couple model of the membrane. The membrane is assumed to consist of two laterally incompressible leaflets which are in close contact but unconnected. Shapes are determined by minimizing the membrane bending energy at a given volume of a cell (V), given average membrane area (A) and given difference of the areas of two leaflets (A). Different classes of shapes exist in parts of the v/a phase diagram, where v and a are the volume and the leaflet area difference relative to the sphere with area A. The limiting shapes are composed of sections of spheres with only two values allowed for their radii. Two low energy axisymmetrical classes, which include discocyte and stomatocyte shapes are studied and their phase diagrams are analyzed. For v=0.6, the discocyte is the lowest energy shape, which transforms by decreasing a continuously into a stomatocyte. The spontaneous membrane curvature (C ₀) and compressibility of membrane leaflest can be incorporated into the model.A model, where A is free and C ₀ determines the shapes at given V and A, is also studied. In this case, by decreasing C ₀, a discocyte transforms discontinuously into an almost closed stomatocyte.     

Does acetylcholine change the electrical resistance of the basal membrane of secretory cells in eccrine sweat glands?

Summary The present experiment was intended to study whether or not acetylcholine decreases the electrical resistance of the basal membrane of secretory cells in stimulating eccrine secretion of fluid and electrolytes. An isolated segment of the secretory coil of the monkey palm eccrine sweat gland was dissected outin vitro and immobilized in the tip of a constriction pipette. Using a bridge-balanced single glass microelectrode, input impedance of the secretory cell was compared before and after local superfusion of acetylcholine in each cell. The mean input impedance was 27m, which did not significantly change after application of acetylcholine. Between 15 and 30 sec after cessation of acetylcholine superfusion, input impedance increased by 42% and then returned to normal within 60 sec. The current-induced voltage deflection due to intraluminally injected current pulse was measured across both the basal membrane (V _b ) and the epithelial wall (V _t ) as qualitative measures of the respective membrane resistances. Both V _b and V _t increased by about 10%, but their ratio remained unchanged after stimulation with acetylcholine. A Ca⁺⁺ ionophore, A23187, which is as potent a stimulant of eccrine sweat secretion as acetylcholinein vitro, also failed to change the above two parameters. It was concluded that the decrease in the electrical resistance of the basal membrane of the secretory cells could not be detected in the sweat gland after stimulation with acetylcholine or A23187. The possibility was discussed that the action of acetylcholine at the basal membrane is one of enhancing the activity of the nonconductive pathway rather than the conductive pathway in this exocrine gland.     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.     

Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti

Two-electrode voltage clamp (TEVC) methods were used to explore conductive transport pathways in principal cells, the dominant cell type in Malpighian tubules of the yellow fever mosquito. The basolateral membrane of principal cells had a voltage (V_bl) of -85.1 mV in 49 principal cells under control conditions. Measures of the input resistance R_pc together with membrane fractional resistance yielded estimates of the conductance of the basolateral membrane (g_bl = 1.48 μS) and the apical membrane (g_a = 3.13 μS). K⁺ channels blocked by barium accounted for 0.94 μS of g_bl. Estimates of transference numbers yielded the basolateral membrane Na⁺ conductance of 0.24 μS, leaving 0.30 μS (20%) of g_bl unaccounted. The secretagogue db-cAMP (0.1 mM), a known activator of the basolateral membrane Na⁺ conductance, significantly depolarized V_bl to -65.0 mV and significantly increased g_bl from 1.48 μS to 2.47 μS. The increase was blocked with amiloride (1 mM), a known blocker of epithelial Na⁺ transport. The inhibition of metabolism with di-nitrophenol significantly depolarized V_bl to -9.7 mV and significantly increased R_pc from 391.6 kΩ to 2612.5 kΩ. Similar results were obtained with cyanide, but it remains unclear whether the large increases in R_pc stem from the uncoupling of epithelial cells and/or the shutdown of conductive transport pathways in basolateral and apical membranes. Our results indicate that the apical membrane of principal cells is more than twice as conductive as the basolateral membrane. Partial ionic conductances suggest the rate-limiting step for transepithelial Na⁺ secretion at the basolateral membrane.     

Phosphate uptake inLemna gibba G1: energetics and kinetics

Phosphate uptake was studied by determining [³²P]phosphate influx and by measurements of the electrical membrane potential in duckweed (Lemna gibba L.). Phosphate-induced membrane depolarization (E _m ) was controlled by the intracellular phosphate content, thus maximal E _m by 1 mM H₂PO ₄ ^- was up to 133 mV after 15d of phosphate starvation. The E _m was strongly dependent on the extracellular pH, with a sharp optimum at pH 5.7. It is suggested that phosphate uptake is energized by the electrochemical proton gradient, proceeding by a 2H⁺/H₂PO ₄ ^- contransport mechanism. This is supported also by the fusicoccin stimulation of phosphate influx. Kinetics of phosphate influx and of E _m , which represent mere plasmalemma transport, are best described by two Michaelis-Menten terms without any linear components.Abbreviations E _m electrical membrane potential difference - E _m phosphate-induced, maximal membrane depolarization - FW fresh weight     

Active and passive properties of rabbit descending colon: A microelectrode and nystatin study

Summary The electrical properties of the basolateral membrane of rabbit descending colon were studied with microelectrode methods in conjunction with the polyene antibiotic nystatin. Two problems were examined: (i) the relative distribution of tight junctional, apical membrane and basolateral membrane resistances, and (ii) the ionic basis of the basolateral membrane potential. Intracellular K⁺ activity (K⁺) was measured using liquid ion exchanger microelectrodes ((K⁺)=76±2mm) and was found not to be in equilibrium with the basolateral membrane potential. In order to measure membrane resistances and to estimate the selective permeability of the basolateral membrane, the apical membrane was treated with nystatin and bathed with a K₂SO₄ Ringer's solution which was designed to mimic intracellular K⁺ composition. This procedure virtually eliminated the resistance and electromotive force of the apical membrane. Shunt resistance was calculated by two independent methods based on microelectrode and transepithelial measurements. Both methods produced similar results (R _s =691±63 cm² and 770±247 cm², respectively). These findings indicate that the shunt has no significant selectivity, contrary to previous reports. Native apical membrane resistance was estimated as 705±123 V cm² and basolateral membrane resistance was 95±14 V cm².To estimate basolateral membrane selectivity, the serosa was bathed in a NaCl Ringer's solution followed by a series of changes in which all or part of the Na⁺ was replaced by equimolar amounts of K⁺. From measures of bi-ionic potentials and conductance during these replacements, we calculated potassium permeability and selectivity ratios for the nystatin-treated colon by fitting these results to the constant field equations. By correcting for shunt conductance, it was then possible to estimate the selective permeability of the basolateral membrane alone. Selectivity estimates were as follows:P _Na/P _K=.08 andP _Cl/P _K=.07 (uncorrected for shunt) andP _Na/P _K=.04 andP _Cl/P _K=.06 (basolateral membrane alone).In a second set of experiments, evidence for an electrogenic Na⁺ pump in the basolateral membrane is presented. A small ouabain-sensitive potential could be generated in the nystatin-treated colon in the absence of chemical or electrical gradients by mucosal, but not serosal, addition of NaCl. We conclude that this electrogenic pump may contribute to the basolateral membrane potential; however, the primary source of this potential is passive: specifically, a potassium gradient which is maintained by an active transport process.An appendix compares the results of nystatin experiments to amiloride experiments which were conducted separately on the same tissues. The purpose of this comparison was to develop a comprehensive model of colonic transport. The analysis reveals a leak conductance in the apical membrane and the presence of an amiloride-insensitive conductance pathway.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Electrolytes control flows of water across the apical barrier in toad skin: The hydrosmotic salt effect

Summary The reversible dependence of skin osmotic water permeability (L _PD ) upon the ionic concentration of the outer bathing solution — which we have called hydrosmotic salt effect (HSE) — was studied in the isolated skin of the toadBufo marinus ictericus. The skin osmotic water flow (J _V ) was measured as a function of outer bathing solution osmolality (O _e ).L _PD , calculated as (J _v /) _P=0 (where and P are the osmotic and hydrostatic pressure differences across the skin, respectively) was constant whenO _e was altered with sucrose, a nonelectrolyte. In contrast,L _PD increased continuously in the hypotonic range asO _e was raised from zero (distilled water) with NaCl or KCl. The HSE could also be evoked in the condition of reversed osmotic volume flow, with the outer bathing medium made hypertonic with sucrose.Diffusional¹⁴C-sucrose permeability, measured in theJ _v =0 condition to prevent solvent drag of sucrose in the paracellular pathways, indicate that the hydrosmotic salt effect cannot be explained by assuming a paracellular permeability increase, due to tight junction opening, but might be interpreted as due to changes in the osmotic water permeability of the apical membranes of the most superficial cells of the epithelium.The hydrosmotic salt effect can be elicited in control skins and in vasopressin-stimulated skins, on top of the hormonal response.The time course of the hydrosmotic salt effect is substantially different from that of the hydrosmotic response to vasopressin. Its half-time is 4 to 5 times faster than that of vasopressin action, with individual values as short as 1.5 min.The time courses of the hydrosmotic salt-effect onset and reversibility are exponential, clearly contrasting with the typical sigmoidal shape of vasopressin onset and washout time courses.Based on time course data and on speed of response we postulate that the mechanism underlying the hydrosmotic salt effect is due to modifications of existing water pathways in the apical membrane, rather than to incorporation and removal of water permeability units in this structure.     

Basolateral membrane potassium conductance of A6 cells

Summary To study the properties of the basolateral membrane conductance of an amphibian epithelial cell line, we have adapted the technique of apical membrane selective permeabilization (Wills, N.K., Lewis, S.A., Eaton, D.C., 1979b, J. Membrane Biol. 45:81–108). Monolayers of A6 cells cultured on permeable supports were exposed to amphotericin B. The apical membrane was effectively permeabilized, while the high electrical resistance of the tight junctions and the ionic selectivity of the basolateral membrane were preserved. Thus the transepithelial current-voltage relation reflected mostly the properties of the basolateral membrane. Under basal conditions, the basolateral membrane conductance was inward rectifying, highly sensitive to barium but not to quinidine. After the induction of cell swelling either by adding chloride to the apical solution or by lowering the osmolarity of the basolateral solution, a large out-ward-rectifying K⁺ conductance was observed, and addition of barium or quinidine to the basolateral side inhibited, respectively, 82.4±1.9% and 90.9±1.0% of the transepithelial current at 0 mV. Barium block was voltage dependent; the half-inhibition constant (K _i) varied from 1499±97 m at 0 mV to 5.7±0.5 m at –120 mV.Cell swelling induces a large quinidine-sensitive K⁺ conductance, changing the inward-rectifying basolateral membrane conductance observed under basal conditions into a conductance with outward-rectifying properties.     

Electrical membrane potential and resistance in photoautotrophic suspension cells of Chenopodium rubrum L.

On photoautotrophically grown, suspension-cultured cells of Chenopodium rubrum L. the electrical potential difference V _mand the electrical resistance across plasmalemma and tonoplast have been measured using one or two intracellular micro-electrodes. In a mineral test-medium of 5.8 mM ionic strength V _mvalues between 100 and 250 mV, 40% thereof between 170 and 200 mV, and a mean value (±S.E.M.) of 180.6±3.4 mV have been recorded. The average membrane input resistance R _mwas 269±36 M, corresponding to an average membrane resistivity r _mof 3.0 m². V _mand r _mare sensitive to light, temperature, and addition of cyanide, suggesting the presence of an electrogenic hyperpolarizing ion pump, and are ascribed essentially to the plasmalemma. A hexose-specific saturable electrogenic membrane channel is identified through a decrease of V _mand r _mupon addition of hexoses. The hexoseconcentration-dependent depolarization V _msaturates at 92 mV and returns half-saturating concentrations (apparent k _mvalues) of 0.16 mM galactose, 0.28 mM glucose, and 0.48 mM fructose. The magnitude of V _mand r _mwell agrees with pertinent data from mesophyll cells in situ (where only V _mdata are available) and from photoautotrophic lower plant cells. However, V _mis markedly higher than reported for heterotrophically grown suspension cells of different higher plants (with which r _mdata have not been reported so far). It is concluded from the present study and a companion paper on water transport (Büchner et al., Planta, in press) that photoautotrophically grown Chenopodium suspension cells closely resemble mesophyll cells as to cell membrane transport properties.Abbreviations V _m membrane potential(mV) - R _o input resistance () - R _m membrane input resistance () - r _m specific resistance (resistivity) of the membrane (m²)     

Retention of basic electrophysiologic properties by human sweat duct cells in primary culture

Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V _a ) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na⁺−K⁺ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na⁺ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (V_a) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na⁺ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture. Cl⁻ substitution at the apical side by the impermeant anion gluconate depolarized the V _a of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized (ΔV _a =−8.3±1.2mV,n=5) or depolarized (ΔV _a =8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest that CD cells, like MPD cells, retain significant Na⁺ and Cl⁻ conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared to MPD cells. This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation.     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

The mechanism of Na+ transport by rabbit urinary bladder

Summary The mechanism of Na⁺ transport in rabbit urinary bladder has been studied by microelectrode techniques. Of the three layers of epithelium, the apical layer contains virtually all the transepithelial resistance. There is radial cell-to-cell coupling within this layer, but there is no detectable transverse coupling between layers. Cell coupling is apparently interrupted by intracellular injection of depolarizing current. The cell interiors are electrically negative to the bathing solutions, but the apical membrane of the apical layer depolarizes with increasingI _sc. Voltage scanning detects no current sinks at the cell junctions or elsewhere. The voltage-divider ratio, , (ratio of resistance of apical cell membrane,R _a, to basolateral cell membrane,R _b) decreases from 30 to 0.5 with increasingI _sc, because of the transportrelated conductance pathway in the apical membrane. Changes in effective transepithelial capacitance withI _sc are predicted and possibly observed. The transepithelial resistance,R _t, has been resolved intoR _a, R_b, and the junctional resistance,R _j, by four different methods: cable analysis, resistance of uncoupled cells, measurements of pairs of (R _t, ) values in the same bladder at different transport rates, and the relation betweenR _t andI _sc and between andI _sc.R _j proves to be effectively infinite (nominally 300 k F) and independent ofI _sc, andR _a decreases from 154 to 4 k F with increasingI _sc. In the resulting model of Na⁺ transport in tight epithelia, the apical membrane contains an amiloride-inhibited and Ca⁺⁺-inhibited conductance pathway for Na⁺ entry; the basolateral membrane contains a Na⁺–K⁺-activated ATPase that extrudes Na⁺; intracellular (Na⁺) may exert negative feedback on apical membrane conductance; and aldosterone acts to stimulate Na⁺ entry at the apical membrane via the amiloride-sensitive pathway.     

The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Intracellular potassium activity in mammalian proximal tubule: Effect of perturbations in transepithelial sodium transport

Summary Intracellular potassium activity (a _{K i} ) was measured in control conditions in mid-cortical rabbit proximal convoluted tubule using two methods: (i) by determination of the K⁺ equilibrium potential (E _K) using Ba²⁺-induced variations in the basolateral membrane potential (V _BL) during transepithelial current injections and (ii) with double-barrel K-selective microelectrodes. Using the first method, the meanV _BL was –48.5±3.2 mV (n=16) and the meanE _K was –78.4±4.1 mV corresponding to aa _{K i} of 68.7mm. With K-selective microelectrodes,V _BL was –36.6±1.1 mV (n=19),E _K was –64.0±1.1 mV anda _{K i} averaged 40.6±1.7mm. While these lastE _K andV _BL values are significantly lower than the corresponding values obtained with the first method (P<0.001 andP<0.01, respectively), the electrochemical driving force for K transport across the basolateral membrane ( _K =V _BL –E _K) is not significantly different for both techniques (30.1±3.3 mV for the first technique and 27.6±1.8 mV for ion-selective electrodes). This suggests an adequate functioning of the selective barrel but an underestimation ofV _BL by the reference barrel of the double-barrel microelectrode. Such double-barrel microelectrodes were used to measure temporal changes ina _{K i} and _K in different experimental conditions where Na reabsorption rate (J _Na) was reduced.a _{K i} was shown to increase by 12.2±2.7 (n=5) and 14.1±4.4mm (n=5), respectively, whenJ _Na was reduced by omitting in the luminal perfusate: (i) 5.5mm glucose and 6mm alanine and (ii) glucose, alanine, other Na-cotransported solutes and 110mm Na. In terms of the electrochemical driving force for K exit across the basolateral membrane, _K, a decrease of 5.4±2.0 mV (P<0.05,n=5) was measured when glucose and alanine were omitted in the luminal perfusate while _K remained unchanged whenJ _Na was more severely reduced (mean change =–1.7±2.1 mV, NS,n=5). In the latter case, this means that the electrochemical driving force for K efflux across the basolateral membrane has not changed while both the active influx through the Na–K pump and the passive efflux in steady state are certainly reduced. If the main pathway for K transport is through the basolateral K conductance, this implies that this conductance must have decreased in the same proportion as that of the reduction in the Na–K pump activity.